The invention relates to amplification of nucleic acids using biochemical amplification products, and in particular how to carry out such amplification in an automated fashion using robotic colony pickers and well plates.
As is well known, sequencing methods such as gel capillary electrophoresis require large numbers of copies of the genetic material of interest. It is therefore necessary to provide an efficient amplification process for replicating a small sample of the genetic material of interest.
Traditional amplification processes are based on inserting a sample of the nucleic acid of interest, e.g. a DNA or RNA fragment, into bacterial plasmids or artificial chromosomes, and then returning the recombinant material thus obtained into a bacterial or yeast host. Bacterium division or yeast division is then promoted by growing colonies of the bacteria or yeast in culture to provide the desired amplification, i.e. multiple copying. A typical culturing time is 24 hours. Following amplification, the samples are purified prior to sequencing.
More recently, biochemical amplification products have become available, for example the enzyme from bacteriophage phi29, marketed under the trade name “TempliPhi” by Amersham Biosciences. Biochemical amplification products include ones based on rolling circle amplification (RCA), such as bacteriophage phi29, and ones based on polymerase chain reaction (PCR), such as Taq polymerase or other thermostable DNA polymerase.
In principle, biochemical amplification offers several advantages over amplification in a bacterial or yeast host.
Firstly, the amplification process, being an essentially chemical rather than biological process, can be made quicker, since it is not dependent on culturing. For example, DNA sequences can be determined directly from bacterial colonies avoiding the necessity for growing colonies in culture.
Secondly, the amplified sample can be directly sequenced without the need for an intermediate purification step.
However, at least some biochemical amplification products are not compatible with colony picking robots because of contamination problems. Contamination is a specific problem with biochemical amplification products, because they are extremely sensitive to non-specific amplification. To understand the contamination problem when using colony picking robots, the basic operation of a colony picking robot is briefly described.
A colony picking robot uses a pin head comprising an array of pins each of which can be “fired” pneumatically, where firing is used to describe the pneumatically driven extension and retraction of each pin to and from a deployed position. The pins are arranged in an array matched to standard well plate dimensions. For example a 12×8 pin head is common with the pins laid out in a square grid matched to a 96-well well plate. The pin head is movable over the bed of the robot using high precision xyz-positioners. An individual colony is picked by moving the picking head over the colony of interest, which may be contained in a culture dish or tray clamped to the bed of the robot, so that an as-yet unused pin is over the colony. The pin is then fired down to touch the colony and thereby pick up sample from the colony. The pin is then retracted. The pin head is then moved to a well plate in which the sample is to be deposited. The pin carrying the sample is arranged over the well of the well plate chosen to receive that sample and the pin fired once again so that the pin head is immersed in solution contained in the target well (i.e. buffer), thereby releasing the sample into the well (i.e. inoculating).
Once all the pins have been used, and hence are “dirty”, the pin head is moved to a wash station for cleaning. The wash station will have one or more baths. For example a wash station may have a single ethanol bath. Another example would be to have two baths, one containing ethanol and the other a mixture of bleach and distilled water. All pins of the pin head are fired into the cleaning liquid and washed. Washing may be assisted by mounting the bath on a shaker.
After washing and subsequent drying, the pins on the pin head are clean and available for further colony picking and inoculation.
With traditional amplification in a bacterial or yeast host, which is essentially a biological process, it is only necessary that any bacterium or yeast host on the pin heads is killed in the cleaning step performed at the wash station. Provided that the host is killed, it cannot divide, so amplification cannot occur. In the context of a picking robot, this means that, even if dead host material is left on a pin head after cleaning, contamination of a sample in another well by adding some of the dead host material from a previous picking event into it will not harm the process, since the contaminant material cannot be amplified. Amplification of a contaminant is referred to as non-specific amplification. In practice it has been found that when using colony picking robots non-specific amplification can be avoided with bacterial or yeast hosts without difficulty by thorough washing.
On the other hand, with biochemical amplification, which is essentially a chemical process, what is relevant is that the pin heads do not carry any chemically active residue from a previous sample after cleaning. Moving from a biological to a chemical process therefore imposes much more stringent requirement on the pin cleaning. In particular, it has been found that it is not feasible to carry out processes with the (bio)chemical amplification product bacteriophage phi29 in a colony picking robot with standard washing protocols, because of the cross-contamination and resultant non-specific amplification.